The present invention provides new triazolo[4,5-d]pyrimidine compounds, their use as medicaments, compositions containing them and processes for their preparation.
Platelet adhesion and aggregation are initiating events in arterial thrombosis. Although the process of platelet adhesion to the sub-endothelial surface may have an important role to play in the repair of damaged vessel walls, the platelet aggregation that this initiates can precipitate acute thrombotic occlusion of vital vascular beds, leading to events with high morbidity such as myocardial infarction and unstable angina. The success of interventions used to prevent or alleviate these conditions, such as thrombolysis and angioplasty is also compromised by platelet mediated occlusion or re-occlusion.
A number of converging pathways lead to platelet aggregation. Whatever the initial stimulus, the final common event is a cross linking of platelets by binding of fibrinogen to a membrane binding site, glycoprotein IIb/IIIa (GPIIb/IIIa). The high anti-platelet efficacy of antibodies or antagonists for GPIIb/IIIa is explained by their interference with this final common event. However, this efficacy may also explain the bleeding problems that have been observed with this class of agent. Thrombin can produce platelet aggregation largely independently of other pathways but substantial quantities of thrombin are unlikely to be present without prior activation of platelets by other mechanisms. Thrombin inhibitors such as hirudin are highly effective anti-thrombotic agents, but again may produce excessive bleeding because they function as both anti-platelet and anti-coagulant agents (The TIMI 9a Investigators (1994), Circulation 90, pp. 1624-1630; The Global Use of Strategies to Open Occluded Coronary Arteries (GUSTO) IIa Investigators (1994) Circulation 90, pp. 1631-1637; Neuhaus K. L. et. al. (1994) Circulation 90, pp.1638-1642).
It has been found that ADP acts as a key mediator of thrombosis. A pivotal role for ADP is supported by the fact that other agents, such as adrenaline and 5-hydroxytryptamine (5HT, serotonin) will only produce aggregation in the presence of ADP. The limited anti-thrombotic efficacy of aspirin may reflect the fact that it blocks only one source of ADP which is that released in a thromboxane-dependent manner following platelet adhesion (see e.g. Antiplatelet Trialists"" Collaboration (1994), Br. Med. J. 308, pp. 81-106; Antiplatelet Trialists"" Collaboration (1994), Br. Med. J. 308, pp.159-168). Aspirin has no effect on aggregation produced by other sources of ADP, such as damaged cells or ADP released under conditions of turbulent blood flow. ADP-induced platelet aggregation is mediated by the P2T-receptor subtype uniquely located on the platelet membrane. Recently it has been shown that antagonists at this receptor offer significant improvements over other anti-thrombotic agents. Accordingly there is a need to find P2T-antagonists as anti-thrombotic agents.
It has now been found that a series of triazolo[4,5-d]pyrimidine derivatives are P2T-receptor antagonists. In a first aspect the invention therefore provides a compound of formula (I): 
wherein:
R1 is a C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8-cycloalkyl, aryl or a thienyl group, each group being optionally substituted by one or more substituents selected from halogen, OR8, NR9R10, SR11 or C1-6 alkyl (itself optionally substituted by one or more halogen atoms); R2 is C1-8 alkyl, C2-8 alkenyl or C3-8 cycloalkyl each of which may be optionally substituted by one or more substituents selected from halogen, OR8, NR9R10, SR11, C1-6 alkyl, C3-8 cycloalkyl, pyridyl or aryl (the latter two of which may be optionally substituted by one or more substitutents selected from halogen, OR20, C(O)R11, NR14C(O)R15, NR16SO2R17, SO2NR18R19, nitro, NR12R13, SR11, methylenedioxy or C1-6 alkyl which is optionally substituted by one or more halogen atoms);
R3 and R4 are both hydroxy;
R5 is hydrogen or C1-6 alkyl; R6 is C1-6 alkyl, optionally substituted by one or more groups selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, halogen, OR21, C3-6 cycloalkyl, or R6 is C3-6 cycloalkyl, or R6 is xe2x80x94A-phenyl or xe2x80x94A-pyridyl each of which may be optionally substituted by one or more groups selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, halogen, OC1-6 alkyl, C3-6 cycloalkyl, or methylenedioxy; or R5 R6 together with the nitrogen atom to which they are attached for a 5 to 7-membered saturated ring optionally substituted by C1-6 alkyl;
A is a bond or C1-6 alkyl;
R8 is hydrogen, C1-6 alkyl (which may be optionally substituted by one or more halogen atoms) or aryl (which may be optionally substituted by one or more substituents selected from halogen, nitro, C(O)R11, OR20, SR11, NR12R13, NR14C(O)R15, NR16SO2R17, SO2NR18R19);
R9 is hydrogen, C1-6 alkyl (which may be optionally substituted by one or more halogen atoms) or aryl (which may be optionally substituted by one or more substituents selected from halogen, nitro, C(O)R20, OR20, SR11, NR12R13, NR14C(O)R15, NR16SO2R17, SO2NR18R19);
R10 is hydrogen, C1-6 alkyl or C(O)C1-6 alkyl;
R12 and R13 are independently hydrogen, C1-6 alkyl or together with the nitrogen atom to which they are attached form a 4- to 8-membered ring;
R15 is C1-6 alkyl or phenyl;
R11, R14 and R16 are independently hydrogen or C1-6 alkyl;
R17 is C1-6 alkyl or phenyl;
R18 and R19 are independently hydrogen, C1-6 alkyl or phenyl;
R20 is hydrogen, phenyl or C1-6 alkyl (which may be optionally substituted by halogen);
R21 is hydrogen or C1-6 alkyl, provided that when R21 is H, R5 must be C1-6 alkyl. or a pharmaceutically acceptable salt or solvate thereof.
Alkyl groups, whether alone or as part of another group, can be straight chained or branched. Aryl groups include phenyl and naphthyl groups. Acyl groups include C(O)C1-6alkyl such as acetyl and 1-oxopropyl.
Suitably R1 is C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8-cycloalkyl, aryl or a thienyl group, each group being optionally substituted by one or more substituents selected from halogen, OR8, NR9R10, SR11 or C1-6 alkyl (itself optionally substituted by one or more halogen atoms). Preferably R1 is C1-6 alkyl, thienyl, or trifluoromethylphenyl. More preferably R1 is methyl, propyl, propenyl or thienyl.
Suitably R2 is C1-8 alkyl, C2-8 alkenyl or C3-8 cycloalkyl each of which may be optionally substituted by one or more substituents selected from halogen, OR8, NR9R10, SR11, C1-6 alkyl, C3-8 cycloalkyl, pyridyl or aryl (the latter two of which may be optionally substituted by one or more substituents selected from halogen, OR20, C(O)R11, NR14C(O)R15, NR16SO2R17, SO2NR18R19, nitro, NR12R13, SR11, methylenedioxy or C1-6 alkyl which is optionally substituted by one or more halogen atoms); where R8, R9, R10, R11, R14, R15, R16, R17, R18 and R19 are as defined above. Preferably R2 is C1-6 alkyl or a C3-8-cycloalkyl group optionally substituted by phenyl which itself can be optionally substituted by halogen, OR8 or C1-6-alkyl. More preferably R2 is cyclopropyl optionally substituted by phenyl, 4-methoxyphenyl, 3-chlorophenyl, 4-methylphenyl or 4-fluorophenyl.
Suitably R5 is hydrogen or C1-6 alkyl; R6 is C1-6 alkyl, optionally substituted by one or more groups selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, halogen, OR21, C3-6 cycloalkyl, or R6 is C3-6 cycloalkyl, or R6 is -A-phenyl or -A-pyridyl each of which may be optionally substituted by one or more groups selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, halogen, OC1-6 alkyl, C3-6 cycloalkyl, or methylenedioxy; or R5 and R6 together with the nitrogen atom to which they are attached for a 5 to 7-membered saturated ring optionally substituted by C1-6 alkyl. Preferably R5 is hydrogen and R6 is cyclopropyl, methylcyclopropyl, C1-6 alkyl, C1-6 alkyl substituted by halogen, preferably one or more flouoro atoms. More preferably R5 is ethyl, isopropyl or isobutyl, or R5 and R6 together with the nitrogen atom to which they are attached form a pyrrolidine ring. Most preferably R5 is hydrogen and R6 is methyl or 2-fluoroethyl.
Particularly preferred compounds of the invention include those exemplified herein, both in free base form and as a pharmaceutically acceptable salt or solvate thereof.
According to the invention there is further provided a process for the preparation of a compound of formula (I) which comprises reaction of a compound of formula (II): 
where R1 and R2 are as defined in formula (I) or are protected derivatives thereof, P1 and P2 are hydrogen or protecting groups and Y is CO2H, with a compound of formula (III):
R5R6NHxe2x80x83xe2x80x83(III)
where R5 and R6 are as defined in formula (I), and optionally thereafter in any order:
converting one or more functional groups into a further functional groups
removing any protecting groups
forming a pharmaceutically acceptable salt or solvate.
Examples of suitable groups which each P1 and P2 can independently represent are C1-6-alkyl (preferably methyl), benzyl, (C1-6-alkyl)3Si (preferably trimethylsilyl) and a C(O)C1-6-alkyl group (preferably acetyl). Preferably the two groups P1 and P2 together with the atoms to which they are attached complete a ring, for example the two groups P1 and P2 together represent an alkylidene such as methylidene or, more preferably, isopropylidene, or an alkoxy methylidene such as ethoxymethylidene.
The reaction of compounds of formula (II) and (III) is preferably carried out in the presence of a coupling agent using methods known from peptide synthesis (see M. Bodanszky and A. Bodanszky, The Practice of Peptide Synthesis, Springer-Verlag, 1984). Suitable coupling agents include 1,1xe2x80x2-carbonyldiimidazole and dicyclohexylcarbodiimide; the preferred coupling agent is bromo-tris-pyrrolidino-phosphonium hexafluorophosphate or benzotriazole-1-yl-oxy-tris-(dimethylamino)phosphoniumhexafluorophosphate, used in the presence of N,N-diiisopropylethylamine. The reaction is preferably carried out in N,N-dimethylformamide (DMF) or tetrahydrofuran THF) and preferably at a temperature of from xe2x88x9215xc2x0 to 120xc2x0 C., more preferably at a temperature of from 0xc2x0 C. to room temperature.
Protecting groups can be added and removed using known reaction conditions. The use of protecting groups is fully described in xe2x80x98Protective Groups in Organic Chemistryxe2x80x99, edited by J W F McOmie, Plenum Press (1973), and xe2x80x98Protective Groups in Organic Synthesisxe2x80x99, 2nd edition, T W Greene and P G M Wutz, Wiley-Interscience (1991).
Alternative methods of activating a compound of formula (II) wherein Y is CO2H include formation of an acyl halide or an acetic anhydride. Acid anhydrides may be formed by treatment with an acyl halide, such as acetyl chloride in the presence of a base, such as pyridine or by treatment with a dehydrating agent such as acetic acid anhydride or phosphorus pentoxide in an inert solvent. Acyl halides may be formed by treatment of the acid with a halogenating agent, for example P(III), P(V) or S(IV) halides such as phosphorus trichloride. Acyl halides may also be prepared by an exchange reaction of the acid with an acyl halide such as oxalyl bromide. The reactions may be performed in the halogenating agent or acyl halide as solvent or in other inert solvents such as methylene chloride, at a temperature of from 0 to 150xc2x0 C. Activation is preferably carried out by treatment with oxalyl chloride in dichloromethane at room temperature.
Deprotection can be carried out using methods generally known in the art. For example for groups P1/P2 deprotection is preferably carried out as follows:
(i) where one or both of P1 and P2 represent C(O)C1-6-alkyl, these groups can be removed by basic hydrolysis, for example by using a metal hydroxide, preferably an alkali metal hydroxide, such as sodium hydroxide or lithium hydroxide, or quaternary ammonium hydroxide in a solvent, such as aqueous ethanol or aqueous tetrahydrofuran, at a temperature of from 10xc2x0 to 100xc2x0 C., preferably the temperature is around room temperature; or by acidic hydrolysis using a mineral acid such as HCl or a strong organic acid such as trichloroacetic acid in a solvent such as aqueous 1,4-dioxane;
(ii) where one or both of P1 and P2 represent (C1-6-alkyl)3Si, these can be removed by the use of, for example, a fluoride ion source, for example tetra-n-butylammonium fluoride or hydrogen fluoride;
(iii) where one or both of P1 and p2 represent a C1-6-alkyl group, these groups can be removed by the use of, for example, boron tribromide;
(iv) where one or both of P1 and p2 represent a benzyl group these can be removed by hydrogenolysis using a transition metal catalyst, for example palladium on charcoal, under an atmosphere of hydrogen, at a pressure of from 1 to 5 bar, in a solvent, such as acetic acid; and/or
(v) where both P1 and P2 together represent alkylidene or an alkoxy alkylidene, they can be removed by the use of, for example, a mineral or organic acid, preferably by using 2M aqueous hydrochloric acid in methanol/1,4-dioxane at room temperature.
Compounds of formula (III) are commercially available or can be prepared by literature methods known to those skilled in the art.
A compound of formula (II) wherein Y is CO2H, CONR5R6 or CO2Rxe2x80x2 can be prepared by reacting a compound of formula (IV): 
wherein R1, P1, P2 R5 and R6 are as defined above, L1 is a leaving group, for example a halogen atom and Rxe2x80x2 is a C1-6-alkyl or benzyl group, with an amine NH2R2 or a salt of NH2R2 wherein R2 is as defined above, in the presence of a base. Suitable salts of NH2R2 include hydrochlorides. Suitable bases include an organic base such as triethylamine or an inorganic base such as potassium carbonate. The amines NH2R2 can be prepared using procedures described in H Nishiyama etal, Bull. Chem. Soc., Jpn., 1995, 68, 1247, P. Newman, Optical Resolution Procedures for Chemical Compounds, Vol. 1, Amines and Related Compounds; Optical Resolution and Information Centre: Manhattan College, Riverdale, N.Y., 1978, p120, J. Vallgarda etal, J. Chem. Soc. Perkin 1, 1994, 461. Certain amines NH2R2 are novel compounds and form a further aspect of the invention.
A compound of formula (IV) can be prepared by diazotising a compound of formula (V): 
wherein R1, Y, L1, P1 and P2 are as defined above, with a metal nitrite, for example an alkali metal nitrite, especially sodium nitrite in dilute aqueous acid, for example 2M HCl, or with a C1-6-alkyl nitrite in an inert solvent, at a temperature of from xe2x88x9220 to 100xc2x0 C.; preferred conditions are isoamyl nitrite in acetonitrile at 80xc2x0 C.
A compound of formula (V) where Y is CO2H can be prepared by reducing and hydrolysing a compound of formula (VI): 
wherein R1, L1, P1 and P2 are as defined above. The reduction may be carried for example by using hydrogenation with a transition metal catalyst at a temperature around room temperature, for example palladium on charcoal under an atmosphere of hydrogen, preferably at a pressure from 1 to 5 atmospheres, in a solvent, for example ethanol, or by using iron in an acidic solvent such as acetic acid at a temperature of about 100xc2x0 C.
To prepare a compound of formula (V) wherein Y is CO2H, following the above reaction, hydrolysis of the compound derived from the compound of formula (VI) may be performed by using a mineral acid such as HCl or a strong organic acid such as trifluoroacetic acid in a solvent such as aqueous 1,4-dioxane, at a temperature of from 20 to 150xc2x0 C. Preferably the reduction and hydrolysis are carried out simultaneously using iron in an acidic solvent for example acetic acid, containing an alkaline earth metal halide, for example calcium chloride, at a temperature of about 80xc2x0 C.
To prepare a compound of formula (V) wherein R9 is C1-6-alkyl or benzyl, the compound of formula (VI) is treated with iron in acetic acid at a temperature of from 50 to 80xc2x0 C. so that the nitro group is reduced. The resulting intermediate is then treated with sodium borohydride in a mixture of water and C1-6-alkyl alcohol or benzyl alcohol at around room temperature.
A compound of formula (VI) can be prepared by reacting a compound of formula (VII): 
wherein L1 and R1 are as defined above and L2 is a leaving group, for example a halogen atom, wherein L1 and L2 are preferably the same, with a compound of formula (VIII): 
wherein P1 is as defined above, in the presence of a base such as C1-6-alkyl-M or MH wherein M is a metal, for example butyl lithium, in an inert solvent, such as tetrahydrofuran (THF), at a temperature of from xe2x88x9210 to 100xc2x0 C. Preferably sodium hydride is used in THF at room temperature.
A compound of formula (VII) may be prepared from 4,6-dihydroxy-2-mercaptopyrimidine by alkylation with R1L3 wherein R1 is as defined above and L3 is a suitable leaving group, for example a halogen atom, followed by nitration, whereafter the two alcohols are is converted to leaving groups L1 and L2.
A compound of formula (V) where Y is CONR5R6 can be prepared by reacting a compound of formula (IX): 
with a compound of formula (VII) where P1, P2, R1, R5, R6, L1, and L2 are as defined above in a suitable solvent such as 1,4-dioxane in the presence of a base such as N,N-diisopropylethylamine followed by reduction of the nitro group. Compounds of formula (IX) may be prepared using procedures described in WO9528160.
The group SR1 can be interconverted by oxidation of the sulphur, for example using oxone(copyright) or mCPBA, followed by treatment with a compound R1SM where R1 is a different R group and M is a metal such as sodium.
All novel intermediates form a further aspect of the invention.
Salts of the compounds of formula (I) may be formed by reacting the free acid, or a salt thereof, or the free base, or a salt or a derivative thereof, with one or more equivalents of the appropriate base (for example ammonium hydroxide optionally substituted by C1-6-alkyl or an alkali metal or alkaline earth metal hydroxide) or acid (for example a hydrohalic (especially HCl), sulphuric, oxalic or phosphoric acid). The reaction may be carried out in a solvent or medium in which the salt is insoluble or in a solvent in which the salt is soluble, e.g. water, ethanol, THF or diethyl ether, which may be removed in vacuo, or by freeze drying. The reaction may also be a metathetical process or it may be carried out on an ion exchange resin. The non-toxic physiologically acceptable salts are preferred, although other salts may be useful, e.g. in isolating or purifying the product.
The compounds of the invention act as P27-receptor antagonists. Accordingly, the compounds are useful in therapy, especially adjunctive therapy, particularly they are indicated for use as: inhibitors of platelet activation, aggregation and degranulation, anti-thrombotic agents or in the treatment or prophylaxis of unstable angina, coronary angioplasty (PTCA), myocardial infarction, perithrombolysis, primary arterial thrombotic complications of atherosclerosis such as thrombotic or embolic stroke, peripheral vascular disease, myocardial infarction with or without thrombolysis, arterial complications due to interventions in atherosclerotic disease such as angioplasty, endarterectomy, stent placement, coronary and other vascular graft surgery, thrombotic complications of surgical or mechanical damage such as tissue salvage following accidental or surgical trauma, reconstructive surgery including skin and muscle flaps, conditions with a diffuse thrombotic/platelet consumption component such as disseminated intravascular coagulation, thrombotic thrombocytopaenic purpura, haemolytic uraemic syndrome, thrombotic complications of septicaemia, adult respiratory distress syndrome, anti-phospholipid syndrome, heparin-induced thrombocytopaenia and pre-eclampsia/eclampsia, or venous thrombosis such as deep vein thrombosis, venoocclusive disease, haematological conditions such as myeloproliferative disease, including thrombocythaemia, sickle cell disease; or in the prevention of mechanically-induced platelet activation in vivo, such as cardiopulmonary bypass and extracorporeal membrane oxygenation (prevention of microthromboembolism), mechanically-induced platelet activation in vitro, such as use in the preservation of blood products, e.g. platelet concentrates, or shunt occlusion such as in renal dialysis and plasmapheresis, thrombosis secondary to vascular damage/inflammation such as vasculitis, arteritis, glomerulonephritis, inflammatory bowel disease and organ graft rejection, conditions such as migraine, Raynaud""s phenomenon, atheromatous plaque formation/progression, vascular stenosis/restenosis and asthma, in which platelet-derived factors are implicated in the disease process.
According to the invention there is further provided the use of a compound according to the invention in the manufacture of a medicament for the treatment of the above disorders. The invention also provides a method of treatment of the above disorders which comprises administering to a patient suffering from such a disorder a therapeutically effective amount of a compound according to the invention.
The compounds may be administered topically, e.g. to the lung and/or the airways, in the form of solutions, suspensions, HFA aerosols and dry powder formulations; or systemically, e.g. by oral administration in the form of tablets, pills, capsules, syrups, powders or granules, or by parenteral administration in the form of sterile parenteral solutions or suspensions, by subcutaneous administration, or by rectal administration in the form of suppositories or transdermally.
The compounds of the invention may be administered on their own or as a pharmaceutical composition comprising the compound of the invention in combination with a pharmaceutically acceptable diluent, adjuvant or carrier. Particularly preferred are compositions not containing material capable of causing an adverse, e.g. an allergic, reaction.
Dry powder formulations and pressurized HFA aerosols of the compounds of the invention may be administered by oral or nasal inhalation. For inhalation the compound is desireably finely divided.
The compounds of the invention may also be administered by means of a dry powder inhaler. The inhaler may be a single or a multi dose inhaler, and may be a breath actuated dry powder inhaler.
One possibility is to mix the finely divided compound with a carrier substance, e.g. a mono-, di- or polysaccharide, a sugar alcohol or another polyol. Suitable carriers include sugars and starch. Alternatively the finely divided compound may be coated by another substance. The powder mixture may also be dispensed into hard gelatine capsules, each containing the desired dose of the active compound.
Another possibility is to process the finely divided powder into spheres which break up during the inhalation procedure. This spheronized powder may be filled into the drug reservoir of a multidose inhaler, e.g. that known as the Turbuhaler(copyright) in which a dosing unit meters the desired dose which is then inhaled by the patient. With this system the active compound with or without a carrier substance is delivered to the patient.
The pharmaceutical composition comprising the compound of the invention may conveniently be tablets, pills, capsules, syrups, powders or granules for oral administration; sterile parenteral or subcutaneous solutions, suspensions for parenteral administration or suppositories for rectal administration.
For oral administration the active compound may be admixed with an adjuvant or a carrier, e.g. lactose, saccharose, sorbitol, mannitol, starches such as potato starch, corn starch or amylopectin, cellulose derivatives, a binder such as gelatine or polyvinylpyrrolidone, and a lubricant such as magnesium stearate, calcium stearate, polyethylene glycol, waxes, paraffin, and the like, and then compressed into tablets. If coated tablets are required, the cores, prepared as described above, may be coated with a concentrated sugar solution which may contain e.g. gum arabic, gelatine, talcum, titanium dioxide, and the like. Alternatively, the tablet may be coated with a suitable polymer dissolved in either a readily volatile organic solvent or an aqueous solvent.
For the preparation of soft gelatine capsules, the compound may be admixed with e.g. a vegetable oil or polyethylene glycol. Hard gelatine capsules may contain granules of the compound using either the above mentioned excipients for tablets, e.g. lactose, saccharose, sorbitol , mannitol, starches, cellulose derivatives or gelatine. Also liquid or semisolid formulations of the drug may be filled into hard gelatine capsules.
Liquid preparations for oral application may be in the form of syrups or suspensions, for example solutions containing the compound, the balance being sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally such liquid preparations may contain colouring agents, flavouring agents, saccharine and carboxymethylcellulose as a thickening agent or other excipients known to those skilled in art.
The invention is illustrated by the following examples. In the examples the NMR spectra were measured on a Varian Unity Inova 300 or 400 spectrometer and the MS spectra were measured as follows: EI spectra were obtained on a VG 70-250S or Finnigan Mat Incos-XL spectrometer, APCI spectra were obtained on Finnigan Mat SSQ7000 or a Micromass Platform spectrometer. Preparative HPLC separations were generally performed using a Novapak(copyright), Bondapak(copyright) or Hypersil(copyright) column packed with BDSC-18 reverse phase silica. Flash chromatography (indicated in the Examples as (SiO2)) was carried out using Fisher Matrix silica, 35-70 xcexcm.
For examples which show the presence of rotamers in proton NMR spectra only the chemical shifts of the major rotamer are quoted.